Abstract
ADP-glucose pyrophosphorylase (AGPase) catalyzes a rate-limiting step in glycogen and starch synthesis in bacteria and plants, respectively. Plant AGPase consists of two large and two small subunits that were derived by gene duplication. AGPase large subunits have functionally diverged, leading to different kinetic and allosteric properties. Amino acid changes that could account for these differences were identified previously by evolutionary analysis. In this study, these large subunit residues were mapped onto a modeled structure of the maize (Zea mays) endosperm enzyme. Surprisingly, of 29 amino acids identified via evolutionary considerations, 17 were located at subunit interfaces. Fourteen of the 29 amino acids were mutagenized in the maize endosperm large subunit (SHRUNKEN-2 [SH2]), and resulting variants were expressed in Escherichia coli with the maize endosperm small subunit (BT2). Comparisons of the amount of glycogen produced in E. coli, and the kinetic and allosteric properties of the variants with wild-type SH2/BT2, indicate that 11 variants differ from the wild type in enzyme properties or in vivo glycogen level. More interestingly, six of nine residues located at subunit interfaces exhibit altered allosteric properties. These results indicate that the interfaces between the large and small subunits are important for the allosteric properties of AGPase, and changes at these interfaces contribute to AGPase functional specialization. Our results also demonstrate that evolutionary analysis can greatly facilitate enzyme structure-function analyses.
Highlights
ADP-glucose pyrophosphorylase (AGPase) catalyzes a rate-limiting step in glycogen and starch synthesis in bacteria and plants, respectively
We identified amino acid residues that were conserved in one large subunit group but not conserved in another large subunit group and amino acid residues that are conserved within large subunit groups but are variable among large subunit groups
We identified 21 type II sites (Fig. 1; Supplemental Table S1) by doing an analysis of all of the pairwise comparisons between the different large subunit groups shown in Supplemental Figure S1 (Georgelis et al, 2008)
Summary
ADP-glucose pyrophosphorylase (AGPase) catalyzes a rate-limiting step in glycogen and starch synthesis in bacteria and plants, respectively. AGPase large subunits have functionally diverged, leading to different kinetic and allosteric properties. Amino acid changes that could account for these differences were identified previously by evolutionary analysis In this study, these large subunit residues were mapped onto a modeled structure of the maize (Zea mays) endosperm enzyme. This possibly reflects the different 3-PGA, G-1-P, and ATP levels in the various tissues This evidence indicates that did the different large subunit groups subfunctionalize in terms of expression, and these groups may have specialized in terms of protein function. While the study of Crevillen et al (2003) pointed to functional specialization of the large subunit, the identity of the amino acid sites in the large subunit that account for these kinetic and allosteric differences was not pursued
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